Annals of Oncology Advance Access published January 10, 2005

Annals of Oncology Advance Access published January 10, 2005 Original article Annals of Oncology doi:10.1093/annonc/mdi077 Expression of survivin and bax/bcl-2 in peroxisome proliferator activated receptor-g ligands induces apoptosis on human myeloid leukemia cells in vitro J. J. Liu 1, R. W. Huang 1 *, D. J. Lin 1, J. Peng 2,X.Y.Wu 1, Q. Lin 1, X. L. Pan 2, Y. Q. Song 3, M. H. Zhang 2, M. Hou 2 & F. Chen 2 1 Department of Hematology and Oncology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou; 2 Department of Hematology and Oncology, Qilu Hospital of Shandong University, Jinan; 3 Department of Oncology, Health Science Center of Peiking University, Beijing, P.R. China Received 24 August 2004; revised 26 September 2004; accepted 5 October 2004 Introduction The present study was undertaken to investigate the mechanisms of peroxisome proliferator activated receptor-g (PPAR-g) ligand-induced apoptosis on human myeloid leukemia K562 and HL-60 cell lines. The results revealed that both 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ) have significant anti-proliferation- and apoptosis-inducing effects on these two kinds of leukemia cells. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly using Wright s and Hoechst 33258 staining. Reverse transcription PCR and western blot analyses demonstrated that both survivin and bcl-2 expression were downregulated markedly, while bax expression was upregulated concurrently when apoptosis occurred. We therefore conclude that 15d-PGJ2 and TGZ have significant apoptosis effects on K562 and HL-60 cells in vitro, and that upregulation of bax as well as downregulation of survivin and bcl-2 expression may be the important apoptosis-inducing mechanisms. The results suggest that PPAR-g ligands may serve as potential therapeutic agents for both acute and chronic myeloid leukemia. Key words: apoptosis, bax, bcl-2, PPAR-g, survivin Peroxisome proliferator activated receptor-g (PPAR-g) ligands have recently been demonstrated to affect proliferation, differentiation and apoptosis of different cancer cell types [1]. In this study, we investigated the expression of PPAR-g in human leukemia K562 and HL-60 cell lines and detected the expressions of survivin and bax/bcl-2 in PPAR-g ligands troglitazone (TGZ)- and 15-deoxy-delta(12,14)-prostaglandin J2 (l5d-pgj2)-induced apoptosis on the two cell lines. Materials and methods Main reagents TGZ and l5d-pgj2 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The following antibodies were purchased from Santa Cruz Company (Heidelberg, Germany): anti-ppar-g, anti-survivin, anti-bcl-2 and anti-bax. TRIZOL reagent was from Gibco (Life Technologies, Rockville, MD, USA), and the reverse transcriptional kit was from MBI (San Francisco, CA, USA). The PCR primers were purchased from Shanghai Shenggong Company (Shanghai, China). Cell culture and cell viability assay K562 and HL-60 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and l00 mg/ml streptomycin, in a humidified 5% CO 2 incubator at 378C. Cells in logarithmic growth phase were used for further experiments. Cell viability was assessed by MTT assay. Briefly, cells at 1 10 5 /ml were treated with various concentrations of PPAR-g ligands (TGZ: 5, 10, 20, 40, 60, 80 and 100 mmol/l; l5d-pgj2: 1, 5, 10, 20, 30, 40 and 50 mmol/l) in 96-well plates for 72 h. Then, MTT working solution was added to each well and cells were incubated for 4 h. The water-insoluble formazan was formed during incubation and was solubilized by adding solubilization agent to each well. The amount of formazan was determined measuring the absorbance at 540 nm using an ELISA plate reader. Flow cytometry detection *Correspondence to: Professor R.W. Huang, The Third Affiliated Hospital of Sun Yat-sen University, Tianhe Road 600, Guangdong, Guangzhou, P.R.China. Tel: +86-20-85516867-2227; Fax: +86-20-87536401; E-mail: huangrw56@163.com For flow cytometry (FCM) analysis, cells treated with TGZ and l5d-pgj2 for 72 h were collected, pelleted, washed with phosphate-buffered saline (PBS) and resuspended in PBS containing 20 mg/l propidium iodide (PI) and 1 g/l ribonuclease A (RNase A). Fixed cells (1 10 6 ) were examined per experimental condition by flow cytometry. The percentage of degraded q 2005 European Society for Medical Oncology

Page 2 of 5 Table 1. PCR primers used in this study PPAR-g mrna (474 bp) Sense primer: 5 0 -TCTCTCCGTAATGGAAGACC-3 0 Anti-sense primer: 5 0 -GCATTATGAGACATCCCCAC-3 0 b-actin mrna (243 bp) Sense primer: 5 0 -CTTCTACAATGAGCTGCGTG-3 0 Anti-sense primer: 5 0 -TCATGAGGTAGTCAGTCAGG-3 0 Survivin mrna (393 bp) Sense primer: 5 0 -CTTTCTCAAGGACCACCGCATC-3 0 Anti-sense primer: 5 0 -CAATCCATGGCAGCCAGCTGC-3 0 bcl-2 mrna (478 bp) Sense primer: 5 0 -CTACGAGTGGGATGCGGGAGATG-3 0 Anti-sense primer: 5 0 -GGTTCAGGTACTCAGTCATCCACAG-3 0 bax mrna (258 bp) Sense primer: 5 0 -ACCAAGAAGCTGAGCGAGTGTC-3 0 Anti-sense primer: 5 0 -TGTCCAGCCCATGATGGTTC-3 0 PPAR-g, peroxisome proliferator activated receptor-g. DNA was determined by the number of cells displaying subdiploid (sub-g 1 ) DNA divided by the total number of cells examined. Detection of mrna expressions using reverse transcription PCR Reverse transcription (RT) PCR was used to detect the expression of PPAR-g as well as survivin and bcl-2/bax before and after the cells were treated with TGZ and l5d-pgj2 for 72 h. The total RNA was extracted using TRIZOL reagent. First-stranded cdna was synthesized using 5 mg total RNA by RT PCR kit. The related PCR primers listed in Table 1 were used to produce the respective correlated products. The PCRs for PPAR-g and b-actin cdnas were performed with 30 amplification cycles and the reaction conditions were: denaturation at 948C for 1 min, annealing at 538C for 2 min and extension at 728C for 3 min. The PCRs for both survivin and bcl-2/bax cdnas were performed with 40 amplification cycles each as follows: (i) survivin and bcl-2: denaturation at 948C for 45 s, annealing at 618C for 1 min and extension at 728C for 1 min; (ii) bax: denaturation at 948C for 45 s, annealing at 588C for 1 min and extension at 728C for 1 min. All of the above PCRs were incubated in an automatic heat-block (Model PJ 2000 DNA Thermal Cycler; Perkin-Elmer, NJ, USA). The PCR products were then run on 1.5% agarose gel and visualized by ethidium bomide staining. Hoechst 33258 staining After the cells were treated with 15d-PGJ2 (40 mmol/l) and TGZ (80 mmol/l) for 72 h, cells were harvested and fixed with 4% formaldehyde in PBS for 10 min, stained with Hoechst 33258, and then subjected to fluorescence microscopy. Wright s staining The morphological variations such as apoptosis, as well as maturation and differentiation, were observed after the cells were treated with 40 mmol/l 15d-PGJ2 for 72 h. DNA fragmentation assay Cells (2 10 6 ) were immersed in cytolysis buffer and incubated for 3 h at 508C. DNA was extracted with phenol chloroform, precipitated in 1/10 volume of 2 M NaAc and two volumes of ethanol overnight, recovered by centrifugation, and resuspended in TE buffer. RNase A was then added (200 mg/l) and treated for 30 min, and then electrophoresed on a 1.2% agarose gel. Western blot analysis Cells (2 10 6 ) were washed twice with ice-cold PBS and lysed for 30 min at 48C; debris was then removed by centrifugation and equivalent amounts of protein were separated by 10% SDS PAGE and transferred onto nitrocellulose filter. The filters were first stained to confirm uniform transfer of all samples and then incubated in blocking solution for 2 h at room temperature. The filters were reacted first with the following antibodies: anti- PPAR-g, anti-survivin, anti-bcl and anti-bax at a dilution of 1:1000 for 2 h, followed by two extensive washes with PBS twice and Tris-buffered saline, ph 8.0, with 0.1% Tween (TBST). Filters were then incubated with horseradish peroxidase-conjugated secondary antibodies of 1:1000 for 1 h, washed with TBST and developed using the Super Signal West Pico Kit (Super Signal West Pico, Pierce, Rockford, IL, USA). As an internal control, b-actin was detected with anti-b-actin antibodies. Statistical analysis All experiments were performed in triplicate and the results are expressed as mean ± SD. Statistical analyses were performed by Student s t-test using SAS 6.12 software. Statistical significance was accepted at the level of P <0.05. Figure 1. Peroxisome proliferator activated receptor-g (PPAR-g) expression in HL-60 and K562 cells detected by reverse transcription (RT) PCR and western blot. (A) PPAR-g mrna expression. Lane 1, HL-60 cells; lane 2, K562 cells; lanes 3 and 4, b-actin; lane 5, marker. (B) Western blot analysis of PPAR-g expression in HL-60 and K562 cells.

Page 3 of 5 Figure 2. Cell viability caused by 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ). K562 and HL-60 cells were plated on 96-well plates, and on the second day cells were treated with different concentrations of TGZ and 15d-PGJ2 for 72 h. Experiments were carried out in triplicate. The cell viability was then determined by MTT assay, as described in Materials and methods. Values represent mean (± SD) cell viability as a percentage of untreated control samples. Results PPAR-g expression in the two cell lines In the two myeloid leukemia cell lines, PPAR-g expression was detected by RT PCR and western blot. As shown in Figure 1A (agarose), the fragment of PPAR-g mrna was 474 bp in length, and the internal control, b-actin mrna, was detected (length 243 bp). As shown in Figure 1B, PPAR-g protein expression was observed very clearly in the two kinds of cells using western blot analysis (molecular mass of PPARg protein was 55 kda). Cell viability and FCM analysis As shown in Figure 2, both TGZ and 15d-PGJ2 had significant growth inhibition effects on the two kinds of cells in a dosedependent manner. The two kinds of leukemia cells showed different sensitivity to TGZ and 15d-PGJ2, both K562 and HL-60 cells were more sensitive to 15d-PGJ2 than to TGZ. Figure 3. Cell apoptosis caused by 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ). After K562 and HL-60 cells were treated with different concentrations of TGZ and 15d-PGJ2 for 72 h, flow cytometry (FCM) analysis was used to detect the apoptotic rate. The cells were stained with propidium iodide and then analyzed by FCM; the percentage of sub-g 1 cells in the two kinds of cell lines was increased in a concentration-dependent manner. In particular, when treated with 40 mmol/l 15d-PGJ2, the percentage of sub-g 1 cells of both K562 and HL-60 cells was >50%. The experiments were repeated three times and the results are presented as mean ± SD. To show whether the cell growth inhibition induced by 15d- PGJ2 and TGZ was caused by induction of apoptosis, the cells were stained with PI and analysed by FCM. As shown in Figure 3, along with the enhancement of cell growth inhibition, apoptotic cells gradually increased, and the percentage of sub-g 1 cells of both K562 and HL-60 cells was higher, >50% when treated with 40 mmol/l 15d-PGJ2 for 72 h. Expression of apoptosis related genes The mrna expressions of both survivin and bcl-2 were downregulated while mrna expression of bax was upregulated. The protein levels of survivin and bcl-2 were downregulated and bax protein expression was upregulated concurrently, as examined by western blotting when apoptosis ocurred (Figure 4). Figure 4. Expressions of pro- and anti-apoptotic genes detected by reverse transcription (RT) PCR (left) and western blot (right) before and after cells were treated with with 40 mmol/l 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and 80 mmol/l troglitazone (TGZ) for 72 h. Lanes 1, K562 cells; lanes 2, HL-60 cells. The expressions of both survivin and bcl-2 were downregulated, while expression of bax was upregulated, as observed by both RT PCR and western blot analysis.

Page 4 of 5 Figure 5. Apoptosis observed by Hoechst 33258 staining (200). (1) HL-60 cells; (2) K562 cells. Marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentation were found clearly. Apoptotic cells increased in a dose-dependent manner in the two kinds of leukemia. Cells were treated with different concentrations of 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) for 72 h. (A) Control. (B) 30 mmol/l 15d- PGJ2. (C) 40 mmol/l 15d-PGJ2. Hoechst 33258 and Wright s staining, and DNA fragmentation analysis Marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were seen clearly using Hoechst 33258 staining (Figure 5). Cell apoptosis was also observed using Wright s staining (Figure 6); there was no cell differentiation or maturation in cell morphology during the whole culture period as observed using Wright s staining. Cell apoptosis was confirmed using DNA fragmentation analysis. Incubation of K562 and HL-60 cells with both 40 mmol/l 15d-PGJ2 and 80 mmol/l TGZ for 72 h Figure 6. Wright s staining (100). Cells were treated with 40 mmol/l 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) for 72 h. Cell apoptosis was observed, represented by chromatin condensation and fragmentation of nuclei. No cell maturation and differentiation was found in either (A) K562 or (B) HL-60 cells. Figure 7. DNA fragmentation analysis. Cells were incubated with 40 mmol/l 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or 80 mmol/l troglitazone (TGZ) for 72 h. (A) Control (no reagents were used). (B) 15d-PGJ2-treated cells. (C) TGZ-treated cells. Lanes 1, K562 cells; lanes 2, HL-60 cells; lane M, marker.

elicited a characteristic ladder of DNA fragments representing integer multiples of the internucleosomal DNA length ( 180 200 bp) (Figure 7). Discussion In this study, we found that the PPAR-g ligands 15d-PGJ2 and TGZ could induce apoptosis in myeloid leukemia K562 and HL-60 cells. RT PCR and western blot analysis revealed that both survivin and bcl-2 expressions were downregulated markedly, while bax expression was concurrently upregulated. We concluded that 15d-PGJ2 and TGZ have significant apoptosis effects on myeloid leukemia cells in vitro. The upregulation of bax and downregulation of survivin and bcl-2 expression may be important apoptotic-inducing mechanisms. To our knowledge, this is the first report to clarify the mechanisms of PPAR-g ligand-induced apoptosis on HL-60 and K562 cells. The regulation of apoptotic cell death has a profound effect on the pathogenesis and progression of cancer cells, and antiapoptotic signals are important in the development and prognosis of hematological malignancies [2, 3]. Survivin, one member of inhibitor of apoptosis family, has recently been reported to play an important role in both cell proliferation and cell death [4]. Downregulation of survivin expression may cause a cell-cycle defect that leads to programmed cell death [4]. bcl-2 and bax are two members of the bcl-2 family, which play different roles in programmed cell death [5]. When bax was overexpressed in cells, apoptotic death in response to death signals was accelerated, leading to its designation as a death agonist [6], and when bcl-2 was overexpressed it heterodimerized with bax and death was repressed. Therefore, the ratio of bcl-2 to bax is important in determining susceptibility to apoptosis [5]. In summary, the results of this study suggest that PPAR-g ligands have significant apoptosis effects on K562 and HL-60 cells in vitro. Upregulation of bax and downregulation of survivin and bcl-2 expression may be the important apoptoticinducing mechanisms. Acknowledgements We thank the members of our laboratories for their insight and technical support. This work is supported by grants from the National Natural Science Foundation of China (No. 30300312). References Page 5 of 5 1. Theocharisa S, Margeli A, Kouraklis G. Peroxisome proliferator activated receptor-g ligands as potent antineoplastic agents. Curr Med Chem Anti-Cancer Agents 2003; 3: 239 251. 2. Rossi D, Gaidano G. Messengers of cell death: apoptotic signaling in health and disease. Haematologica 2003; 88: 212 218. 3. Maggio E, Van den Berg A, Diepstra A et al. Chemokines, cytokines and their receptors in Hodgkin s lymphoma cell lines and tissues. Ann Oncol 2002; 13 (Suppl 1): 52 56. 4. Paydas S, Tanriverdi K, Yavuz S et al. Survivin and aven: two distinct antiapoptotic signals in acute leukemias. Ann Oncol 2003; 14: 1045 1050. 5. Kirkin V, Joos S, Zornig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 2004; 1644: 229 249. 6. Sultana H, Kigawa J, Kanamori Y et al. Chemosensitivity and p53-bax pathway-mediated apoptosis in patients with uterine cervical cancer. Ann Oncol 2003; 14: 214 219.